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- EMDB-35046: Focused map on the aCTD-AfsR-DNA region of the Streptomyces coeli... -

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Basic information

Entry
Database: EMDB / ID: EMD-35046
TitleFocused map on the aCTD-AfsR-DNA region of the Streptomyces coelicolor AfsR-dependent transcription activation complex
Map data
Sample
  • Complex: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
KeywordsRNA polymerase / SARP regulator / gene regulation
Biological speciesStreptomyces coelicolor A3(2) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsWang Y / Zheng J
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31770068, 32070040 China
CitationJournal: PLoS Biol / Year: 2024
Title: Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.
Authors: Yiqun Wang / Xu Yang / Feng Yu / Zixin Deng / Shuangjun Lin / Jianting Zheng /
Abstract: Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide- ...Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.
History
DepositionDec 27, 2022-
Header (metadata) releaseJan 3, 2024-
Map releaseJan 3, 2024-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35046.png

Downloads & links

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Map

FileDownload / File: emd_35046.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-1.264915 - 1.6807703
Average (Standard dev.)0.00030542316 (±0.012900882)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35046_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_35046_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_35046_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of AfsR-dependent transcription activation comp...

EntireName: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
Components
  • Complex: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter

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Supramolecule #1: Cryo-EM structure of AfsR-dependent transcription activation comp...

SupramoleculeName: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#11
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 560 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTRISTRIS
50.0 mMKClKCl
3.0 mMDTTDTT
5.0 mMMgCl2MgCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 193644
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 95223
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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